The present invention relates to a device for performing an assay, which device comprises a substrate having oriented through-going channels, said channels opening out on a surface for sample application, the channels in at least one area of the surface for sample application being provided with a first binding substance capable of binding to an analyte.
Such a device is disclosed in WO95/11755 for xe2x80x9csequencing by hybridizationxe2x80x9d applications. The device comprises a substrate provided with channels, the channels being oriented substantially perpendicular to the surface of the substrate. Three types of substrate are disclosed. The first type is comprised of a multitude of hollow glass fibers. It is manufactured by stacking glass fibers having an etchable core, providing the stack with flat ends, polishing those ends, and etching the cores, usually with acid. The second type of substrate is produced by electrochemical etching of a crystalline silicon wafer. First, the position of the channels as well as their size are defined using standard photolithographic methods. Subsequently the oriented channels are formed electrochemically. The third type of substrate is produced by nuclear track etching of an inorganic substrate. This method, comprising the steps of exposing the substrate to heavy, energetic charged particles and wet-etching, results in a substrate with channels scattered randomly over the surface of the substrate. With higher pore densities and porosity there is more chance of fusion of channels, which show reduced flow resistance with respect to other, non-fused channels.
All three types of substrates are quite expensive because of the labor-intensive manufacturing processes and/or expensive starting materials and wasteful operations, such as sawing and polishing, and/or expensive equipment. In addition, the substrates are characterized by a relatively low porosity of 30% and less. More advantageous, higher porosities of up to 80% are said to be achievable, but only at relatively low channel densities, with the disadvantage that the effective surface area of the channels of a particular area of the substrate is lower in comparison with a substrate having a comparable porosity but with higher channel densities (and consequently narrower channels). The silicon-based substrates as disclosed in WO 95/11755 are not transparent for light. These substrates therefore deteriorate the advantageous use of optical marker systems for the detection of analyte bound in the substrate. Popular optical marker systems are for instance based on enzymatically induced color reactions, on bio- or chemi-luminescence, or on photoluminescence. In the latter case both the excitation light and emitted luminescent light have to pass through the substrate material.
Another device comprising a substrate with through-going oriented channels is described in co-pending application number EP98104938, the contents of which are herewith incorporated by reference.
In EP98/04938 a device is disclosed wherein the porous substrate is an electrochemically manufactured metal oxide membrane.
Metal oxide membranes having through-going, oriented channels can be manufactured cheaply through electrochemical etching of a metal sheet. Metals considered are, among others, tantalum, titanium, and aluminium, as well as alloys of two or more metals and doped metals and alloys. The metal oxide membranes are transparent, especially if wet, which allows for assays using various optical techniques. Such membranes have oriented channels with well controlled diameter and advantageous chemical surface properties. When used in an assay the channels in at least one area of the surface of the electrochemically manufactured metal oxide membrane are provided with a first binding substance capable of binding to an analyte. According to a preferred embodiment the metal oxide membrane is comprised of aluminium oxide.
Therefore aluminium oxide membranes may accommodate for high densities of areas comprising different first binding substances. Aluminium oxide membranes having oriented through-going channels are disclosed by Rigby, W. R. et al. (Trans. Inst. Metal Finish., 68(3), p. 95, 1990).These membranes were used to purify viruses, and to store enzymes for sensor purposes, and, as disclosed in EP98/04938 were found to be highly suitable as substrates in, flow trough devices for, for example, probe-based assays. Reagents used in these assay are immobilized in the channels of the substrate and the sample fluid will be forced trough the channels to be contacted with the reagents.
In WO87/07954 a modification of so called manifold vacuum devices is described: The permeability of the base of the wells in, for example, a 96-well microtiter plate, is used to improve the mixing of fluid in the wells, by repeatedly applying a pressure difference over the porous base of the well and thus forcing the fluid to pass trough the base and, subsequently, back into the well. It was shown that this procedure results in a better mixing of the ingredients (for example, beads, microspheres or other entities of the small fluid sample in the wells) and is thus an alternative for mechanical mixing methods like bubbling, vortexing stirring or agitating the sample fluid by swirling the plate.
For example, as described in WO87/07954 antigen bound glass fibers were formed into a filter and used as a basis in the wells of a manifold plate. An enzyme immunoassay is performed whereby a highly visible purple precipitate is formed that is of large enough size to be trapped by the filter.
The object of the present invention is to provide optimized methods of contacting a flow trough device comprising a substrate having oriented troughgoing channels with the sample fluid. Thus, the present invention provides new and non-obvious ways of performing an assay using flow trough devices with oriented trough going channels in which reagents have been immobilized. Use is made of the capillary forces of the channels in the substrate. Based on the dimensions of the substrate and the device wherein it is used an optimal method of contacting the sample fluid with the reagents in the channels is provided.
With the present invention it has been found that based on the physical parameters of the substrate, the device and the sample volume, a very efficient and sensitive method for analyzing sample fluids can be provided.
Thus, the present invention is concerned with a method for analyzing one or more fluid samples for the presence, amount or identity of one or more analytes optionally present in said samples, said method comprising the steps of:
(a) providing a device comprising one or more round wells with a fixed diameter, said wells exposing a substrate of a specific thickness, said substrate having oriented trough going channels, and the area of the substrate exposed in the well being provided with at least one binding substance specific for at least one of said analytes,
(b) adding an amount of sample fluid to one or more of the wells of the device, the amount of added sample fluid being calculated on the basis of the dimensions of the wells and the substrate,
(c) generating an alternating flow trough the substrate in the wells whereby the liquid volume of sample fluid is forced to pass trough the channels in the substrate from the upper side of the substrate to the lower side of the substrate and back at least one time, under conditions that are favorable to a reaction between any analyte present in the sample and the binding substance(s),
(d) reading any signal generated in any of the wells and
(e) determining from said signal(s) the presence, amount and/or identity of said one or more analytes.
If necessary (for example, in case of an assay where a bound-free separation step is required, the substrate can be washed prior to reading the signal.